1. Field of the Invention
The present invention relates to a reflectron for generating a non-linear retarding electrical field to focus product ions, formed in a time-of-flight mass spectrometer or the like, at the same or substantially the same focal point regardless of mass, to enable a detector, disposed substantially at that focal point, to obtain well resolved mass spectra throughout the product ion mass range without scanning, stepping or otherwise changing the voltage of the reflectron.
2. Description of the Related Art
Mass Spectrometers
Mass spectrometers are used to determine the chemical composition of substances and the structures of molecules. In general, they comprise an ion source where neutral molecules are ionized, a mass analyzer where ions are separated according to their mass/charge ratio, and a detector. Various mass analyzers exist, such as, for example, magnetic field (B) analyzers, combined electrical and magnetic field or double-focusing instruments (EB or BE), quadrupole electric field (Q) analyzers, and time-of-flight (TOF) analyzers.
Tandem Mass Spectrometers
Two or more analyzers may be combined in a single instrument to produce tandem (MS/MS) mass spectrometers. These include triple analyzers (EBE), four sector mass spectrometers (EBEB or BEEB), triple quadrupoles (QqQ) and hybrids such as the EBqQ.
In tandem instruments, the first mass analyzer is generally used to select a precursor ion from among the ions normally observed in a mass spectrum. Fragmentation is then induced in a collision chamber located between the mass analyzers, and the second mass analyzer is used to provide a mass spectrum of the product ions.
Tandem mass spectrometers may be utilized for ion structure studies by establishing the relationship between a series of molecular and fragment precursor ions and their products. More commonly today, they are utilized to determine the structures of components of a compound mixture.
For example, the primary structure of a protein is generally determined by digesting the protein with an enzyme, which cleaves at specific amino acids to produce a set of smaller peptides, separating the resultant peptides by chromatography, and determining the amino acid sequence of each peptide. In many cases, the peptides cannot be separated completely. In such cases, a tandem mass spectrometer employing a soft ionization technique (one which produces molecular ions and a few fragment ions) will record a mass spectrum in the first mass analyzer reflecting the molecular weights of the series of peptides in a particular chromatographic fraction. Each of these can be selected as a precursor ion by the first mass analyzer, fragmented in an intermediate collision chamber, and its product ion spectrum recorded in the second mass analyzer to produce a series of peaks that can be used to elucidate the amino acid sequence.
Sector Instruments
In sector instruments (those utilizing electrostatic analyzers and magnetic fields), it is possible to observe product ion mass spectra on a single (two sector) mass analyzer by scanning the magnetic field and electric field simultaneously. An analogous situation, as described below, occurs for time-of-flight mass spectrometers in that product ions may be observed on a single TOF analyzer equipped with a reflectron, as well as on any number of tandem configurations.
A major limitation on the sensitivity of mass spectrometers arises in instruments which must scan the mass range one mass at a time. For this reason, tandem (four sector) instruments have recently been constructed with spatial array detectors, which record ions over a region (typically 4% to 8%) of the spectrum simultaneously. Spatial array detectors cannot be constructed for quadrupole based instruments, since they do not separate ions by spatial dispersion. Time-of-flight mass analyzers, on the other hand, can record ions over the entire mass range simultaneously.
Time of Flight Mass Spectrometers
Time-of-flight mass spectrometers are relatively simple instruments that record the mass spectra of compounds or mixtures of compounds by measuring the times (usually of the order of tens to hundreds of microseconds) for molecular and/or fragment ions of those compounds to traverse a (generally) field-free drift region. The simplest version of a time-of-flight mass spectrometer, as shown in FIG. 1, consists of a short source region S in which ions are formed and accelerated to their final kinetic energies by an electrical field defined by voltages on the backing plate and drawout grid, a longer field-free drift region D, bounded by the drawout grid and an exit grid, and a detector.
In the most common configuration, the drawout and exit grids (and therefore the entire drift length) are at ground potential, the voltage on the backing plate is V, and the ions are accelerated in the source region to an energy: mv.sup.2 /2=eV, where m=the mass of the ion, v=the velocity of the ion, and e=the charge on an electron. The ions that pass through the drift region and their times of flight as measured by a detector is represented by the following equation: ##EQU1## which shows a square root dependence upon mass. Typically, the source distance S is of the order of 0.5 cm, while drift lengths D ranging from 15 cm to 8 meters have been utilized. Accelerating voltages V generally range from 3 kV to 30 kV, and flight times are of the order of 5 to 100 microseconds.
A time-of-flight mass spectrometer described in 1955 by Wiley and McLaren (Wiley, W. C.; McLaren, I. H.: Review of Scientific Instruments, Vol. 26, No. 12 (1955) pp. 1150-57) resulted in the first commercial instrument that was produced by the Bendix Corporation. In that instrument, volatile compounds were ionized by a pulsed electron beam (electron impact or EI), extracted by a drawout pulse, and accelerated into a one meter flight tube. The entire mass spectrum could be observed as an oscillographic trace triggered by the drawout pulse.
Alternatively, the spectrum could be recorded by measuring the ion current in a short (10-50 ns) window that was incremented in successive time-of-flight cycles that occurred at a repetition rate of 10 kHz. This boxcar or time slice recording method, as taught by Holland et al. (Holland, J. F.; Enke, C. G.; Allison, J.; Stults, J. T.; Pinkston, J. D.; Newcombe, B.; Watson, J. T.: Analytical Chemistry (1983) Vol. 55, 997A) had two major drawbacks. First, it required that the analog transients produced in repetitive cycles be reproducible. This was feasible for the electron impact ionization of gaseous samples, but would not be practical for current methods involving laser induced ionization of non-volatile samples, which generally produce transients which vary widely from laser shot to laser shot. Secondly, the scanning method that was used to reconstruct a mass spectrum did not take advantage of the multiplex recording capability (i.e. the ability to record ions of all masses simultaneously) of the time-of-flight analyzer. This resulted in considerable reduction in duty cycle and loss of ultimate sensitivity that might be required in modern instruments that are used to examine minute quantities of biological samples.
Linear Time-Of-Flight Mass Spectrometers
Linear time-of-flight mass spectrometers generally have low mass resolution (about one part in 300 to 600), that can be attributed, in part, to the initial kinetic energy distribution of the ions. The Wiley-McLaren instrument sought to improve mass resolution using a method known as time-lag focusing. This approach, which utilized a time delay between the ionization and extraction/acceleration events, was unfortunately mass dependent. It was compatible with boxcar recording in that the length of the time delay could be scanned synchronously. It was not, however, compatible with multiplex recording, since only a portion of the mass spectrum would be in focus.
Because the ions have a finite distribution of kinetic energies that results in poor mass resolution, time-of-flight instruments generally incorporate reflectrons which provide a retarding electrical field that reverses the ion trajectories in order to cancel the effects of kinetic energy distribution on ion arrival time at the detector. When used to focus ions that are formed in the ion source, the focusing effects of the reflectron are independent of mass. That is, a detector located at the focal point can be used to record the entire mass range at maximum resolution in each time measurement cycle.
Reflectrons
The reflectron (or ion mirror) is a device for improving the time focusing of groups of ions at the detector, and hence the mass resolution, by compensating for the initial kinetic energy distributions of the ions within the groups of ions, independent of the mass of the ions formed in the ion source. For example, when ions are formed in an ion source and injected into a time-of-flight mass spectrometer system, ions having different mass/charge ratios (e.g. m/z ratios) have essentially the same final kinetic energy after acceleration and thus travel through the flight tube of the time-of-flight mass spectrometer at different speeds. However, the ions may have slightly different initial kinetic energies. Hence, the reflectron is used to "focus" the ions at the same point within the system, with ions of different mass/charge arriving at that point at different times.
To focus the ions, the reflectron, as shown in FIG. 2, generates a retarding electrical field which decelerates the ions essentially to zero velocity, and causes those ions to turn around and return along essentially the same path in the opposite direction. In practice, the return path is at a small angle with respect to the original flight path in order to physically accommodate the reflected ion detector.
As the ions enter the reflectron, ions with higher kinetic energy (velocity) penetrate the reflectron more deeply than those with lower kinetic energy, and thus travel a longer path to their focal point. Hence, a group of ions (e.g. 500 Da) having an initial kinetic energy distribution (i.e. the ions in the group each have slightly different initial kinetic energies) reach the detector with the same initial kinetic energy distribution, but arrive at the detector at essentially the same time.
A single-stage reflectron, as shown in FIG. 3, provides only a single linear retarding field. In contrast, the reflectron described by Mamyrin et al. (Mamyrin, B. A.; Karatajev, V. J.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys. JETP Vol. 37, No. 1 (1973) pp. 45-8) was a dual-stage reflectron. That is: the voltages placed on the stack of ion lenses (as shown in FIG. 2) provided two distinct linear retarding field regions, bounded by grids to minimize field penetration. In this configuration, ions are decelerated to approximately one third of their initial kinetic energies within the first 10% of the reflectron, while the longer second region provides the different path length for ions with different kinetic energies. Thus, a dual-stage configuration enables the reflectron to be much smaller than a single-stage reflectron having an equivalent focal length.
In instruments employing reflectrons, such as time-of-flight mass spectrometers, ions pass through two field-free regions (L.sub.1 and L.sub.2 as shown, for example, in FIG. 2) and into and out of the reflectron, where they turn around at a distance d which is the penetration depth (the depth that the electron penetrates into the reflectron). In instruments incorporating a single-stage reflectron, the total flight time is represented by the following equation: ##EQU2## which follows a square root law similar to that of linear instruments. The focusing action can be understood by replacing the denominator in equation [2] with 2 eV+U.sub.0, where U.sub.0 represents the contribution to the ion velocity from the initial kinetic energy distribution.
Ions with excess kinetic energy spend less time in the linear regions L.sub.1 and L.sub.2 (whose lengths do not change). However, the penetration depth increases for more energetic ions, so that the value of term d increases, with optimal focusing achieved when L.sub.1 +L.sub.2 =4d, that is, when ions spend approximately equal amounts of time in the field-free and reflectron regions (Tang, X; Beavis, R; Ens, W.; Lafortune, F.; Schueler,B.; Standing, K. International Journal of Mass Spectrometry and Ion Processes , Vol. 85 (1988) pp. 43-67). Most importantly, optimal focusing is independent of mass, so that the reflectron voltages, focal point and position of the detector are the same for the entire mass range which can be brought into focus for each time measurement cycle.
Gridless Reflectrons
In addition to the single-stage and dual-stage reflectrons described above, Wollnik et al. (Grix, R.; Kutscher, R.; Li, G.; Gruner, U.; Wollnik, H. Rapid Communications in Mass Spectrometry, Vol. 2, No. 5 (1988) pp. 83-5) have described gridless reflectrons, as shown, for example, in FIG. 4, which are designed to minimize the loss of ion transmission due to collisions of ions with grids. While the potentials placed on successive lens elements differ from those that would be used on gridded reflectrons, the intention is to shape a linear deceleration field while compensating for the field penetration that would occur in the absence of the entrance grid. Thus, the gridless reflectron is essential a linear, single-stage device that can additionally provide spatial refocusing of an initially divergent ion beam.
Coaxial Reflectrons
Coaxial reflectrons, as shown, for example, in FIG. 5, have also been described (Della-Negra, S.: LeBeyec, Y. Anal. Chem. Vol. 57 (1985) pp. 2035-40), which focus a divergent beam onto an annular channel plate detector located at the exit of the ion source. Both single-stage and dual-stage coaxial reflectrons have been designed, consisting of one or two linear retarding field regions, respectively.
Product Ion Mass Spectra
While reflectrons were initially intended to improve mass resolution for molecular and fragment ions formed in the ion source region, they have more recently been exploited for recording the mass spectra of product ions. This is performed by dissociating precursor ions, formed in the source, into product ions.
The product ions are formed outside the ion source region after acceleration by metastable decay, fragmentation induced by collisions with a target gas, collisions with a surface or by photodissociation. Such processes can be represented by the reaction: EQU m.sub.1 .fwdarw.m.sub.2 +n [3]
where m.sub.1, m.sub.2 and n are the masses of the precursor ion, product ion and product neutral, respectively.
The intact precursor ions are recorded at the same flight times as observed in the normal mass spectrum. Product ions have the same velocity as the precursor ions, spend the same time in the field-free regions, and could therefore not be distinguished in instruments without a reflectron. However, because they enter the reflectron with energies equal to (m.sub.2 /m.sub.1)eV, they penetrate the retarding field of the reflectron to a much shallower depth than do the precursor ions, and have total flight time (t.sub.2), corresponding to the equation: ##EQU3## or, in terms of the flight time of the precursor ion (t.sub.1 =t.sub.1 "+t.sub.1 ', where t.sub.1 ' and t.sub.1 " are the times spent in the linear and reflectron regions, respectively), corresponding to the equation: ##EQU4## which shows a linear dependence upon mass. Thus, the reflectron serves as a mass dispersive device for product ions.
Focusing can again be understood by replacing the denominator in equation [4] with eV+U.sub.0. However, (m.sub.2 /m.sub.1)d is smaller than d so that the optimal focal point (where the detector should be positioned, i.e., distance L.sub.2 from the reflectron) will be different for each product ion group. Hence, one could adjust the reflection voltage (for each product ion group) to increase the penetration depth d and achieve optimal focusing for a particular ion group.
Product ions will appear in normal mass spectra as generally weak and poorly-focused peaks which cannot be easily associated with a given precursor ion. However, it is possible to record the product ion mass spectrum for a single precursor, by selecting ions of a single mass for passage through the first drift region. An example of this approach is described by Schlag et al. (Weinkauf, R.; Walter, K.; Weickhardt, C.; Boesl, U.; Schlag, E. W.: Int. J. Mass Spectrom. Ion Processes Vol 44a (1989) pp. 1219-25), in which an electrostatic gate is located in the first drift region. The ions passed by the gate are then fragmented by photodissociation using a pulsed UV laser, and the product ions are detected after reflection.
An alternative approach was introduced by LeBeyec and coworkers using a coaxial dual-stage reflectron, and has been developed by Standing et al. (Standing, K. G.; Beavis, R.; Bollbach, G.; Ens. W.; LaFortune, F.; Main, D.; Schueler, B.; Tang, X; Westmore, J. B. Analytical Instrumentation 16(1) (1987) pp. 173-89) using a single-stage reflectron. In this approach, all ions are permitted to enter the reflectron. A detector is also located at the rear of the reflectron and records neutral species resulting from the metastable decay in the first field-free drift length. Because these neutrals appear at time corresponding to the mass of the precursor ion, it is then possible to only register ions in the reflectron detector when a neutral corresponding to the precursor mass is received. The resultant spectrum, known as a correlated reflex spectrum, can only be obtained from methods which employ single ion pulse counting.
A major limitation of the reflections designed to date is that focusing of product ions (mass resolution) is not constant over the mass range. Specifically, the selected precursor ion mass is generally the most well focused ion in the product ion mass spectrum, while focusing decreases for product ions with lower mass. This is generally attributed to the fact that lower mass product ions do not penetrate the reflectron to as great a depth as ions whose masses are close to the precursor ion mass. Thus, it has been a common observation that lowering the reflection voltages permits recording of the low mass portion of the spectrum with considerably better focus, while the higher mass ions simply pass through the back end of the reflectron.
For this reason, several investigators have suggested stepping the reflectron voltages to record different regions of the mass spectrum, or scanning the reflectron voltages and reconstructing a focused mass spectrum from a series of transients (Weinkauf, R.; Walter, K.; Weickhardt, C.; Boesl, U.; Schlag, E. W. Int. J. Mass Spectrom. Ion Processes Vol. 44a (1989) pp. 1219-25 and Spengler, B.; Kirsch, D.; Kaufmann, R.; Jaeger, E. Rapid Commun. Mass Spectrom. 6 (1992) pp. 105-08). For product ion mass spectra, this approach has the same disadvantages as the time-slice method employed by Wiley and McLaren, in that it does not realize the multiplex recording advantage of the time-of-flight mass spectrometer.
Although product ion mass spectra can be recorded in single TOF analyzers employing a reflectron, a number of investigators have described a variety of tandem configurations in which the first mass analyzer is utilized to select the precursor ion mass, while the second mass analyzer is used to record its product ion mass spectrum. Approaches using two linear TOF mass analyzers (i.e., without reflectrons) and reacceleration of the product ions have been described by Derrick (Jardine, D. R.; Morgan, J.; Alderdice, D. S.; Derrick, P. J.: Org. Mass Spectrom. Vol. 27 (1992) pp. 1077-83) and Cooks (Schey, K. L.; Cooks, R. G.; Grix, R; Wollnik, H., International Journal of Mass Spectrometry and Ion Processes Vol. 77 (1987) pp. 49-61).
A linear/reflectron (TOF/RTOF) configuration has also been reported by Cooks (Schey, K. L.; Cooks, R. G.; Kraft, A.; Grix, R.; Wollnik, H., International Journal of Mass Spectrometry and Ion Processes Vol. 94 (1989) pp. 1-14). Strobel and Russell (Strobel, F. H.; Solouki, T.; White, M. A.; Russell, D. H., J. Am. Soc. Mass Spectrom. Vol. 2 (1990) pp. 91-94); and (Strobel, F. H.; Preston, L. M.; Washburn, K. S.; Russell, D. H., Anal. Chem. Vol. 64 (1992) pp. 754-62) have recently described a hybrid instrument (EB/RTOF) using a double-focusing sector mass analyzer for mass selection and a reflectron TOF to record the product ions.
In addition, Cotter and Cornish (Cornish, T. J.; Cotter, R. J. Analytical Chemistry Vol. 65 (1993) pp. 1043-47) and (Cornish, T. J.; Cotter, R. J. Org. Mass Spectrom. (in press) have described a tandem (RTOF/RTOF) time-of-flight instrument using two reflecting time-of-flight mass analyzers. The first analyzer permits high resolution selection of the precursor ion by electronic gating prior to a collision cell, while the second mass analyzer is used to record the collision induced dissociation (CID) or product ion mass spectrum. In this instrument, both dual-stage and single-stage reflectrons have been used. However, both single and dual stage reflectrons currently used suffer from the focusing limitations described above.
The tandem time-of-flight mass spectrometer has several clear advantages over the reflectron TOF analyzer for recording of product ion mass spectra. In many instances, these advantages resemble the advantages of a four sector (EBEB) instrument over the linked E/B scanning methods employed on two sector (EB) mass spectrometers.
That is, the tandem time-of-flight permits high mass resolution selection of the precursor ion because electronic gating is accomplished as the ions are brought into time focus at the collision chamber. In contrast, ion mass gating in the first linear region (L.sub.1) of a reflectron TOF is carried out prior to focusing by the reflectron. Secondly, the tandem time-of-flight mass spectrometer can more clearly separate metastable process from collision induced dissociation, since metastable ions occurring in the first field free region and traverse the first reflectron do not arrive at the ion mass gate at the same time.